PURPOSE: To compare in vitro the efficacy of five resin-based desensitizing agents at reducing human dentin permeability and to compare their cytotoxicity. The tested hypothesis was that their different curing techniques cause variations in efficiency and cytotoxicity. MATERIALS AND METHODS: Dentin slices (0.5 +/- 0.05 mm thick) were prepared from human third molars (10 per group) and their hydraulic conductance was recorded before and after application of one of the desensitizing agents with a Flodec device. Six desensitizing agents were studied: one light curing agent (Seal and Protect); one self-curing agent (Pain Free); the resin-based agents without any polymerization initiator (Health-Dent, Gluma Desensitizer, Isodan); one oxalate-based agent served as a control (Protect). A MTT assay on L 929 fibroblasts was performed to measure the cytotoxicity of the six desensitizing agents applied onto additional dentin slices (10 per group). RESULTS: All the desensitizing agents resulted in a large decrease in dentin permeability. The best results were obtained with Gluma Desensitizer, Isodan, Pain Free and Protect. A statistically significant difference was found among the materials (P = 0.001). All the materials were non-cytotoxic. Cell viability ranged from 88% for Seal and Protect to 100% for Isodan. No difference was found among their cytotoxicity.